(R)-柠苹酸作为重要聚合物中间体甲基丙烯酸(methacrylic acid,MAA)的化学合成前体,在食品加工、医疗、建筑、照明和家电等领域有着重大的应用前景。(R)-柠苹酸目前主要通过化学法生产,其生物法合成技术仍处于研究阶段。该研究首次在地衣芽孢杆菌中引入异源柠苹酸合酶搭建了(R)-柠苹酸合成途径,以葡萄糖为底物实现了(R)-柠苹酸的合成与积累。进一步建立及优化了重组菌株进行全细胞转化产(R)-柠苹酸的条件,发现温度和时间可以显著影响(R)-柠苹酸的产量。最终,采用CMC培养基制备全细胞催化剂,在37 ℃、pH 7.0、底物质量浓度为100 g/L、菌体浓度OD600值为70、摇床转速为250 r/min的条件下转化120 h后,生成(R)-柠苹酸8.57 g/L,转化率为142.84 mg/g葡萄糖,是目前报道的摇瓶培养合成(R)-柠苹酸的最高水平,为生物法高效合成(R)-柠苹酸的进一步研究提供了参考。
The (R)-citramalate is a chemical precursor for synthesis of methacrylic acid (MAA), which is used in food processing, medical device, construction, lighting, and the home appliance industries. At present, (R)-citramalate is mainly produced by chemical method, and its biological synthesis technology is still in the research stage. In this study, Bacillus licheniformis was used as the chassis cell to successfully construct the (R)-citramalate production pathway for the first time, the heterologous expression of citramalate synthase was realized in the recombinant B. licheniformis and the glucose was used as the substrate to achieve the synthesis and accumulation of (R)-citramalate. Subsequently, the conditions for whole-cell biocatalysis were optimized. It was found that temperature and time could significantly affect the yield of (R)-citramalate. Finally, whole-cell catalysts were prepared in CMC medium, the yield of (R)-citramalate was achieved at 8.57 g/L and the conversion rate of it achieved at 142.84 mg/g glucose after a reaction time of 120 h, by controlling the transformation condition at a pH of 7.0, temperature of 37 ℃, rotation speed of 250 r/min, substrate concentration of 100 g/L and OD600 of 70. To the best of our knowledge, the titer of (R)-citramalate is the highest reported to date in shake flask culture, which provided a reference for further research on efficient biosynthesis of (R)-citramalate.
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